Enhancement of phycocyanobilin biosynthesis in Escherichia coli by strengthening the supply of precursor and artificially self-assembly complex

Phycocyanobilin(PCB)is widely used in healthcare,food processing,and cosmetics.Escherichia coli is the common engineered bacterium used to produce PCB.However,it still suffers from low production level,pre-cursor deficiency,and low catalytic efficiency.In this study,a highly efficient PCB-producing strain was created.First,chassis strains and enzyme sources were screened,and copy numbers were optimized,affording a PCB titer of 9.1 mg/L.Most importantly,the rate-limiting steps of the PCB biosynthetic pathway were determined,and the supply of precursors necessary for PCB synthesis was increased from endogenous sources,affording a titer of 21.4 mg/L.Then,the key enzymes for PCB synthesis,HO1 and PcyA,were assembled into a multi-enzyme complex using the short peptide tag RIAD-RIDD,and 23.5 mg/L of PCB was obtained.Finally,the basic con-ditions for PCB fermentation were initially determined in 250 mL shake flasks and a 5-L bioreactor to obtain higher titers of PCB.The final titer of PCB reached 147.0 mg/L,which is the highest reported titer of PCB so far.This research provided the foundation for the industrial production of PCB and its derivatives.